Marine Biology (2+1)

Staining and Mounting Techniques for Protozoan Ciliates

Staining

The protozoan ciliates after fixation are stained using a 1 % erythrosin solution made by dissolving 1 g of erythrosin (powder) in 99 ml of 5% phenol . During staining this fluid is dropped on specimens previously placed on a membrane filter . This setup is allowed to stand for about 10 minutes. Then the excess stain, if any, is removed by rinsing the specimens on the filter with distilled water until the red colour of excess erythrosin disappears .

Mounting

Before mounting ,the already stained ciliates need to be further processed as follows :

(i) The specimen is first fixed for 2 minutes in champy solution and the fixative drained off (the champy solution is composed of 7 ml of 3% CNN ) potassium dichromate solution , 7 ml of 1% (WN)  chromic oxide or chromic trioxide solution and 4 ml of 2% (YN) osmium tetraoxide solution.

(ii) The specimen is washed twice in Da-Fano solution and rinsed in distilled water . The

Da-Fano solution is composed of 10 ml of buffered concentrated formaldehyde (40%) ,

1 g of cobalt nitrate , 1 g of sodium chloride and 10 ml of distilled water .

iii) The specimen is then placed on a slide and the water drained off.

iv) A mixture of 1 - 2 drops of warm (4 ° C) 10% gelatin solution and 2 drops of sea-water are

added to the specimen. This is mixed and then drained off rapidly before the gelatin sets.

v) The slide is cooled until the gelatin has set . It is then placed in a refrigerator for 5 minutes in

cold 3 % silver nitratesolution .

vi) The slide is rinsed with cold distilled water and is placed in a shallow basin containing distilled water to its 3 cm level .

vii) The slide is then irradiated for 10 - 20 minutes with a UV lamp or longer in sunlight until t he objects have darkened sufficiently.

vii) The animal is finally rinsed with cold distilled water , passed through an alcohol series and mounted.